The present invention relates to concentrated preparations of desmethyl tocopherols, including but not restricted to gamma tocopherol (xcex3T), which localize to lipid environments in cardiovascular tissue and scavenge reactive nitrogen species (RNS) by virtue of a phenolic structural element lacking one or more methyl substituents on the phenolic ring system. The capability to scavenge RNS imparts cardiovascular protective properties to the compound.
Tocopherols are a class of lipophilic, phenolic compounds of plant origin. The major tocopherol found in mammalian tissue is alpha tocopherol (xcex1-tocopherol or xcex1T or vitamin E) FIG. 1, although significant quantities of demethylated (desmethyl) forms (particularly xcex3-tocopherol or xcex3T) FIG. 1, are also present. xcex1-Tocopherol acts as a free radical scavenger (i.e., a chain-breaking antioxidant) when the phenolic head group encounters a free radical:
Toc-OH+L.xe2x86x92Toc-O.+LH Toc-OH=tocopherol L.=lipid radical
The phenoxyl radical Toc-O. is much more stable, and less reactive, than L.. The aromatic nature of the tocopherol ring system, combined with steric and electronic influences from the methyl
The phenoxyl radical Toc-O. is much more stable, and less reactive, than L.. The aromatic nature of the tocopherol ring system, combined with steric and electronic influences from the methyl substituents, stabilizes the tocopheroxyl radical and thereby ends the lipid peroxidation process. Eventually, Toc-O. is reduced back to Toc-OH by ascorbate acting in conjunction with NADPH reductase. While xcex1-tocopherol is the major tocopherol in the body, other tocopherols exist. The second major tocopherol in the human body is xcex3-tocopherol (xcex3T), which, like xcex1-tocopherol, is made by plants and taken into the human diet with foodstuffs.
Recently, it has become appreciated that reactive nitrogen species (RNS) are significant to many diseases including coronary artery disease (CAD), hypertension, and other forms of cardiovascular disease where localized inflammatory reactions occur. RNS are derived from the enzymatic oxidation of arginine via the intermediate nitric oxide free radical (FIG. 2). Unlike oxygen-centered free radicals, reactive nitrogen species are not scavenged effectively by xcex1-tocopherol. On the other hand, xcex3-tocopherol can react easily with RNS because of the presence of an open space on the chromanol head of the molecule (FIG. 1). The major product of xcex3-tocopherol reaction with RNS is 5-nitro-xcex3-tocopherol (5Nxcex3T, FIG. 1). Recent discoveries indicate that: (A) xcex3T protects biological systems from RNS much more effectively than xcex1T; (B) xcex3T is extensively nitrated in human plasma, particularly among smokers and hypertensive individuals; (C) xcex3T inhibits RNS toxicity to a critical enzyme (xcex1-ketoglutarate dehydrogenase, or xcex1KGDH) which is severely damaged in injured vascular tissue; and (D) xcex3T protects cultured endothelial cells from RNS. Thus, xcex3T possesses unique biochemical functions from xcex1T that suggest xcex3T may be a superior dietary supplement, cardioprotectant, cardioplegia additive, or a preservative in cardiovascular tissue exposed to RNS. Other desmethyl tocopherols likewise should be cardioprotective by this mechanism or another.
Chemistry of tocopherol reaction with oxidizing agents. xcex3-Tocopherol is a natural product (a desmethyl tocopherol) of plant origin, present in many vegetable oils, especially soybean oil (1-2). xcex3-Tocopherol is normally taken into the body through consumption of foodstuffs. Human plasma xcex3T concentration is variously reported as 5-30% of xcex1T (3). The xcex3Txcex1T ratio varies markedly among individuals; plasma xcex3T/xcex1T proportionalities may be as low as 0.2% and as high as 30% (inventors"" observations). Both xcex1T and xcex3T are absorbed equally well by the gut, but xcex3T is packaged into lipoproteins less effectively than xcex1T (4). For this reason, xcex1T supplementation decreases systemic xcex3T concentration (3-4).
To date, only three well-disseminated studies have compared xcex1T and xcex3T with respect to their ability to inhibit nitrative stress specifically (5-7). These studies generally investigated the in vitro reaction of nitrating equivalents with target substrates in xe2x80x9cpurexe2x80x9d chemical systems, and two of the three studies reached very different conclusions. The first investigation from Cooney""s lab (5) reported that xcex3T reaction with NO2 gas was 6 times more rapid than the corresponding reaction of xcex1T. Furthermore, exposure of xcex1T (but not xcex3T) to NO2 caused the formation of a secondary nitrating species which could nitrate the target compound morpholine (5). In the same manuscript, Cooney et al. showed that xcex3T was 4-fold more effective than xcex1T at inhibiting neoplastic transformation of methylcholanthrene-treated C3H/10T1/2 fibroblasts, a process which the authors suggest might involve nitrative stress (5). The second study (Christen et al. 1997; reference 6) incorporated either xcex1T or xcex3T, or both, into liposomes which were then exposed to synthetic peroxynitrite (ONOOxe2x88x92). Christen and colleagues found that xcex3T was twice as effective as xcex1T at inhibiting lipid hydroperoxide formation in liposomes exposed to ONOOxe2x88x92. Moreover, these researchers found that xcex3T nitration rates were not influenced by the presence of xcex1T. This latter finding suggests that nitration of xcex3T may occur preferentially to reaction with xcex1T when both tocopherols are simultaneously exposed to a nitrating species. In the third study (7), Goss et al. take issue with the findings of Christen et al. and report that xcex1T does spare xcex3T in liposomes exposed to the superoxide and NO-generating compound SIN-1 [5-amino-2-(4-morpholinyl)-1,2,3-oxadiazolium].
A search of the literature revealed only two studies in which xcex1T and xcex3T were compared for efficacy using in vivo models of cardiovascular stress (no studies were found investigating neurological stress). In the first study (c. 1983), tocopherol-depleted rats were fed xcex1T or xcex3T for two weeks after chronic exposure to iron-dextran as an inducer of oxyradical stress (8). While both xcex1T and xcex3T inhibited systemic lipid oxidation in the animals, xcex3T was approximately 35% as effective as xcex1T. Lipid nitration was not an endpoint of this investigation, and physiologic parameters were not recorded. In a second, very recent study (reference 9; Saldeen et al., J. Am. Coll. Card.,Oct. 1999), rats on an otherwise normal diet were fed xcex1T or xcex3T (100 mg/kg/day) for 10 days after which the abdominal aorta was exposed to patch soaked in 29% FeCl3 (9). This stress induced obstructive thrombus within 20 minutes. Saldeen et al. found that xcex3T supplementation was significantly more effective than xcex1T supplementation at inhibiting iron-induced lipid peroxidation and occlusive thrombus (9). Time to occlusive thrombus was delayed by 25% in the xcex1T supplemented animals while the same parameter was increased by 65% in xcex3T supplemented animals (9). Platelet aggregation kinetics were similarly inhibited, with xcex3T supplementation being 2-fold more efficacious than xcex1T supplementation (9). Most importantly, the xcex3T concentration in the plasma of the xcex3T supplemented rats never exceeded 10% of the xcex1T concentration although the feeding paradigm did increase xcex3T levels 6-fold above baseline (9). By comparison, xcex1T supplementation increased xcex1T plasma concentration only 2-fold (9). When treatment effects were considered in reference to plasma tocopherol concentrations, the Saldeen study found xcex3T to be 20-30 times more potent than xcex1T at inhibition of throbogenic correlates. No conclusive explanation for the xcex3T effect was offered by the Saldeen study, though superoxide dismutase activity increased significantly in the aortas of xcex3T treated animals as compared to the xcex1T treated group (9). The unexpected efficacy of xcex3T might also stem from a differential vascular partitioning of xcex3T, since xcex3T is reportedly incorporated into endothelial cells more rapidly than is xcex1T (10). In any case, the efficacy of xcex3T as a vascular or neuroprotectant cannot be predicted from its bioactivity in traditional fertility assays, or from its oxyradical scavenging capacity as measured in vitro.
Role of oxidative and nitrative stress in atherosclerosis. Oxidative stress is centrally involved with both the initiation and the progression of atherosclerosis. In normal vasculature, low density lipoprotein (LDL) crosses the endothelium to provide lipids and cholesterol to the vascular intima. Normal LDL is taken up in by specific cell-surface receptors whose expression is tightly regulated so as to preclude intracellular accumulation of cholesterol. Chemically-modified LDL, including oxidized LDL (oxLDL), is taken up more rapidly and less specifically, particularly by macrophages (11-22). The accumulation of excessive oxLDL converts these into xe2x80x9cfoam cellsxe2x80x9d, a hallmark of early atherosclerosis (12). Oxidized lipids and proteins are abundant in atherosclerotic lesions, though the specific nature of the oxidative modifications is unclear (13-16). LDL can be oxidized in vitro by exposure to metal-catalyzed oxyradical generating systems, and this oxLDL will convert macrophages into foam cells (13,16); however, this xe2x80x9csyntheticxe2x80x9d oxLDL differs from natural oxLDL in several respects. Synthetic oxLDL is taken up by macrophage scavenger receptors only after complete depletion of xcex1T resident within the LDL particle (18). Natural oxLDL is not recognized by the scavenger receptor, indicating that the chemical modification of natural oxLDL is different from that of in vitro modified LDL (17). Moreover, xcex1T content of native oxLDL is not substantially depleted, even in extracts taken from severe lesions (19). OxLDL is chemotactic and stimulates expression of vascular adhesion molecules, thereby recruiting leukocytes to the subendothelial space (14). Neutrophils and macrophages may become activated in this milieu, releasing pro-inflammatory cytokines and generating more ROS and reactive nitrogen species. Chronic exposure to oxLDL causes macrophage and endothelial death and release of lipids from the dying cells (reviewed in 20). Further leukocyte recruitment to the necrotic focus accelerates the atherogenic process. An additional consequence of subendothelial inflammation is proliferation of VSM cells in response to cytokine exposure, which further decreases perfusion through the affected vessel (20). End-stage disease is characterized by ischemic damage to the heart and major perfused organs, and with increased risk of occlusive thrombus as portions of plaque disintegrate and initiate coagulation cascades.
As previously discussed, the combination of NO with superoxide or other leukocyte-derived oxidants yields peroxynitrite and other nitrating agents. Furthermore, activated macrophages produce profligate quantities of NO via iNOS (inducible nitric oxide synthate). It therefore appears that NO-derived products play a role in vascular modification during atherosclerosis. iNOS and nitrotyrosine have been immunochemically detected in human atherosclerotic plaques, where most staining occurs in foam cells and VSM cells (21-24). Some iNOS is present in VSM even in normal vessel walls (24). Similar iNOS immunoreactivity is found in experimental atheroscerotic lesions of hypercholesterolemic rabbits (25). Endothelial cells express very little iNOS in vivo or in vitro; however, the endothelium is likely to encounter nitrating agents derived from other cell types. Combination of eNOS (endogenous nitric oxide synthate)-derived NO with leukocyte-derived ROS might also form peroxynitrite in the subendothelial space. Quantitative mass spectrometric studies indicate that LDL isolated from human atherosclerotic plaques contains 100 times more nitrotyrosine than LDL from normal plasma (26). Similar LDL protein nitration is observed in rabbits fed a high cholesterol diet (27). Lipid nitration in atherosclerosis has not been investigated.
xcex1-Tocopherol in human cardiovascular disease. Considering the importance of lipoprotein oxidation in the pathogenesis of atherosclerosis, it seems logical that xcex1-tocopherol should decrease the incidence or severity of CAD. In the period from 1985-1995, numerous epidemiological, cross-sectional and observational studies were undertaken to determine if this might be the case. Initial studies using relatively small populations ( less than 100 subjects) failed to find a correlation between xcex1-T and vascular disease, although these studies have been criticized for failure to normalize xcex1T to lipid content, which might confound the interpretation of the data (28). A 1991 study by Gey et al. correlated ischemic heart disease (IHD) rates with lipid-standardized xcex1T concentrations using mean values obtained from male populations in 16 European nations (29). A highly significant negative correlation (copyright)=0.79) was found between these parameters, indicating a beneficial role for xcex1T in IHD. The authors conclude that a 40% increase in plasma xcex1T was associated with an 84% lower mortality rate.
Data from cross-sectional and epidemiological human studies generally support the contention that xcex1T is protective against vascular pathology, though perhaps not in all human populations. In the hope of overcoming limitations inherent to cross-sectional studies, several large-scale, longitudinal investigations were undertaken in the early 1990s to formally test the importance of xcex1-T as a vasoprotectant. The xe2x80x9cUS Nurses"" Health Studyxe2x80x9d analyzed self-reported vitamin E intake among 87,425 American nurses over 8 years (30) and found a 34% diminished risk of coronary disease among subjects within the upper quintile of xcex1T consumption compared to subjects within the lowest quintile. In a similar study (the Health Professions Follow-up Study) involving 39,910 men, the risk of CAD was diminished by 39% for men with a median tocopherol intake of 419 vs. 6.4 IU/day (1 IU=1 mg d-xcex1-tocopherol acetate; 31). From these studies, a daily intake of 100 IU of xcex1T is most consistently associated with benefit (28) while the US Reference Daily Intake for vitamin E is 15 IU/day (34). These several investigations did not discriminate thoroughly between xe2x80x9cdietaryxe2x80x9d versus xe2x80x9csupplementaryxe2x80x9d sources of xcex1T, and no specific consideration was made of xcex3T or other co-antioxidants. In the one large study which has attempted to discriminate between dietary vitamin E and vitamin supplements, 35,000 postmenopausal women were followed for 7 years (the xe2x80x9cWomen""s Health Studyxe2x80x9d, ref. 36). Cardiovascular death was negatively associated with high intake of vitamin E from food, while no benefit was apparent when vitamin E supplements were evaluated alone or in combination with dietary vitamin E intake (36).
Data from large-scale, prospective, controlled tocopherol supplementation trials is currently being analyzed and published with somewhat paradoxical results. Despite epidemiological evidence that xcex1-T correlates inversely with vascular disease, controlled xcex1T supplementation has a relatively subtle protective effect against CAD and possibly a detrimental effect on hemorrhagic pathology. In the Cambridge Heart Antioxidant Study (CHAOS), 2002 male smokers with angiographically proved CAD received vitamin E supplements of 400-800 IU/day and were followed for 18 months (32). In this study, vitamin E supplements caused a significant 77% reduction in nonfatal myocardial infarction but a 29% increase in all-cause mortality. In a similar study involving 1,862 male smokers with previous myocardial infarction, a 50 mg/kg supplement of vitamin E had no effect on MI or mortality after 5.3 years of follow-up (33). An independent study reports that 50 IU/day of xcex1T does not decrease total mortality of smokers but increases death from hemorrhagic stroke after 5-8 years (35). Interestingly, plasma xcex1T increased 50% in this latter supplementation paradigm, a quantity previously associated with an 80% reduction of ischemic heart disease in the cross-cultural epidemiological study by Gey et al. (29). In the most recent evaluation of xcex1T, the Heart Outcomes Prevention Evaluation (HOPE), Canadians at risk for heart disease were studied (38). In a total population of 9541 subjects, 400 IU/day of xe2x80x9cnaturalxe2x80x9d xcex1T had no effect on primary or secondary cardiovascular outcomes or death over a 4.5 year period (38). The quantitative discrepancies between epidemiological data and intervention studies are disturbing. The disparity may indicate that xcex1T can inhibit the development of CAD in the early stages but not in more advanced clinical conditions. Alternatively, it has been suggested that intake of xcex1T from food is correlated with the intake of other co-antioxidants which are required for maximal cardiovascular benefit, and that current xcex1T supplementation paradigms fail to take into account these necessary xe2x80x9ccofactorsxe2x80x9d (36-37). The identity (identities) of these putative xe2x80x9ccofactorsxe2x80x9d has not been suggested.
xcex3-Tocopherol in human biology and cardiovascular disease. Relative to xcex1T, a dearth of epidemiological data exists for xcex3T. Human plasma xcex3T concentration is variously reported as between 5-30% that of xcex1T (41). In platelet-poor plasma, we find that 7% is very close to the correct value in young healthy subjects. The xcex3T/xcex1T ratio varies markedly among individuals; we have observed plasma xcex3T/xcex1T proportionalities as low as 0.2% and as high as 30%. xcex3T is now a major tocopherol in the US diet, due to the high intake of soybean and vegetable oils that are abundant sources of xcex3T (40). Both xcex1T and xcex3T are absorbed equally well by the gut, but xcex3T is packaged into lipoproteins less effectively than xcex1T (39). For this reason, xcex1T supplementation decreases systemic xcex3T concentration (41-42). xcex4-Tocopherol, xcex2-tocopherol and tocol (other demethylated tocopherol homologs) exist in human plasma at approximately 1:10 ratios relative to xcex3T (41). Detailed demographic data regarding plasma and tissue levels of desmethylated tocopherols and their oxidation products have never been published. While extensive data has been collected on xcex1-tocopherol as a possibly beneficial molecule in cardiovascular disease, very little data has been collected on xcex3T.
The small amount of published clinical data regarding xcex3-tocopherol is provocative. Two small studies have investigated xcex3T in CAD. A 1999 study reports a 40% decrease in plasma xcex3T in patients (N=34) with atherosclerosis while xcex1T increased by 30% (43). An earlier 1996 study by Ohrvall et al. found that CAD patients (N=69) had a significant 25% reduction in lipid-normalized plasma xcex3T concentration while xcex1T was statistically unaffected (44). In the latter study, the ratio of xcex3T/xcex1T in the CAD patients was decreased by 35% (44). Importantly, Ohrvall et al. note that very few of the CAD patients had supplemented their diet with vitamin preparations. Tocopherol oxidation and nitration products were not measured in either study. In a separate but very remarkable study of smokers (a group at high risk for vascular disease), plasma xcex3T levels were reduced by more than 50% in chronic smokers while plasma xcex1T concentration was diminished by only 20-25% (45). Moreover, cessation of smoking for 84 hours resulted in a 35% recovery of xcex3T in plasma and a 65% recovery ofxcex3T in low density lipoprotein (LDL) while xcex1T recovery was not significant. Interestingly, the magnitude of xcex3T rebound following cessation of tobacco use correlated very strongly with the extent of tobacco use preceding the period of voluntary abstinence (45). Again, tocopherol oxidation and nitration products were not measured. While several high-profile studies have shown xcex1T intake somewhat protective against CAD in smokers (45), no similar studies have been undertaken using xcex3T as an independent variable.
While chronic xcex1T supplementation can increase plasma levels of xcex1T by 300-400%, very little data exists regarding the effect of dietary xcex3T. To the knowledge of the P.I., no serious attempt has been made to increase plasma xcex3T in humans in the context of a formal scientific study. Several small studies using very small study populations have indicated that dietary supplementation with xcex1T decreases plasma xcex3T in humans and rodents (41-42), while chronic dietary supplementation of xcex3T might conceivably increase plasma and tissue xcex3T concentration. The human biology of other, less common desmethylated tocopherols is essentially uninvestigated. It cannot be assumed, however, that the relative importance of the various tocopherols can be anticipated solely on the basis of their relative tissue concentrations, independent of other biochemical variables.
The present invention is intended to solve the problems described above, namely, the inefficacy of xcex1-tocopherol (vitamin E) to adequately protect against cardiovascular disease in clinical investigations, and to improve the ability of the tocopherol to inhibit the progression of cardiovascular diseases including but not limited to atherosclerosis. The mechanism of the invention at least in part involves the improved ability of a tocopherol desmethyl homolog to scavenge reactive nitrogen species (RNS).
The present invention involves the use of xcex3-tocopherol and other desmethyl tocopherols as scavengers of reactive nitrogen or other reactive species in tissue exposed to an inflammatory stress, particularly in cardiovascular tissue exposed to nitrative stress. The preferred desmethyl tocopherols of the present invention have the following structures: 
The only constraint placed on the structure above is that at least one of the set R1, R2 and R3 must be a H atom. Additionally, the alkyl (linear, branched, or cyclic) tail of the molecule may include either saturated or unsaturated variants (unsaturated variants comprising the chemical subclass of tocotrienol tocopherols). Since the main bioactive function of the above structure is the phenolic head group, any stereoisomer of the tocopherol may be used. Furthermore, since the main bioactive function of the above structure is the phenolic head group, any carbon can be eliminated from the carbon centers labeled 2-4 in the structure above. Furthermore, the xe2x80x94OH group can be esterified or otherwise modified to form a prodrug or a more water-soluble derivative such as an ester, for example, which would regenerate the xe2x80x94OH group in vivo.
These and other homologs of the tocopherols can be chemically synthesized or isolated from natural products. In the method of the present invention, the tocopherols are administered in a safe and effective amount to scavenge reactive nitrogen or other species and slow the progression of nitrative stress in tissue undergoing progressive degeneration. These and other advantages and objects of the invention will be apparent to those skilled in the art.
The present invention also involves a method for protecting or delaying cardiovascular disease, its symptoms, consequences, or related damage. Cardiovascular disease includes ischemia disease (including thrombosis). Mitochondrial function of the myocardial is likewise protected.